Diphenylcyclopropyl analogs as antiestrogenic and antitumor agents

ABSTRACT

Diphenylcyclopropyl analogs in which one or more of the phenyl rings includes substituents comprising a hydroxy group, a hydrogen atom, an acetate group or a substituted or unsubstituted alkoxy group. The compounds are useful as antiestrogens and antitumor agents.

GOVERNMENTAL SUPPORT FOR INVENTION

This invention was made with Government support under a grant from theNational Cancer Institute (CA40458). The Government has certain rightsin this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent ApplicationSer. No. 098,945, filed Sept. 21, 1987, now U.S. Pat. No. 4,879,315which is a continuation-in-part of U.S. patent application Ser. No.363,429 filed Mar. 30, 1982, now abandoned, which is acontinuation-in-part of which U.S. patent application Ser. No. 166,255,filed July 7, 1980, now abandoned, which is a continuation-in-part ofU.S. patent application Ser. No. 128,040, filed Mar. 7, 1980, nowabandoned. U.S. Ser. No. 410,938, filed Sept. 22, 1989, now U.S. Pat.No. 5,015,666 entitled Triarylcyclopropanes as Antiestrogens andAntitumor Agents discloses related subject matter.

These applications are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to diphenylcyclopropyl analog compounds and theiruse in mammals for producing anti-estrogenic activity in the mammal, andfor inhibiting the development of an estrogen-dependent tumor in themammal.

SUMMARY OF THE INVENTION

The present invention comprises a compound having the formula: ##STR1##or any pharmaceutically acceptable salt thereof. X is a halogen orhydrogen. R1 is a hydrogen atom, an acetate group, or a hydroxyl group.R1 may also be an alkoxy group or a substituted alkoxy group in whichthe substituent of the alkoxy group comprises either dialkylamino groupin which the alkyl substitutent contains 1 to 6 carbons, analkylsulfonyloxy group in which the alkyl substituent contains 1 to 6carbons, or a substituted or unsubstituted heterocycle containingbetween about 5 and 6 members. At least one of the members of theheterocycle group is nitrogen. The heterocycle substituent is an alkylgroup containing from 1 to about 6 carbon atoms. The composition mayalso comprise pharmaceutically acceptable salts of the foregoing.

R₂ is a hydrogen atom, an acetate group, or a hydroxyl group. R1 mayalso be an alkoxy group or a substituted alkoxy group in which thesubstituent of the alkoxy group comprises either dialkylamino group inwhich the alkyl substitutent contains 1 to 6 carbons, analkylsulfonyloxy group in which the alkyl substituent contains 1 to 6carbons, or a substituted or unsubstituted heterocycle containingbetween about 5 and 6 members. At least one of the members of theheterocycle group is nitrogen. The heterocycle substituent is an alkylgroup containing from 1 to about 6 carbon atoms. The composition mayalso comprise pharmaceutically acceptable salts of the foregoing.

In the compound of the present invention, R1 and R2 are as describedabove provided that both R1 and R2 cannot be hydrogen, hydroxy, acetateor methoxy.

The compounds of the present invention may be combined with apharmaceutically acceptable carrier to form pharmaceutical compositions.

The present invention further comprises a method of inducingantiestrogenic activity in a mammal in need of such therapy comprisingadministering to the mammal an antiestrogenically effective amount ofone or more compounds having the above-described formula.

The present invention also comprises a method of inhibiting thedevelopment of an estrogen-dependent tumor in a mammal in need of suchtherapy comprising administering to the mammal an effective amount ofone or more compounds having the above-described formula.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention comprises a compound having the formula: ##STR2##or any pharmaceutically acceptable salt thereof.

X represents a halogen or a hydrogen. Preferably X is chlorine.

R₁ represents a hydrogen atom, an acetate group, or a hydroxyl group. R1may also represent an alkoxy group containing from 1 to 3 carbon atoms,and more preferably, from 2 to 3 carbon atoms. R₁ may also be asubstituted alkoxy group in which the alkyl portion thereof contains 1-3carbon atoms and preferably between 2-3 carbons, and in which the alkylportion thereof is substituted with one of the following groups: adialkylamino group, an alkylsulfonyloxy group, or a substituted orunsubstituted heterocycle containing between about 5 and 6 members, oneof which is nitrogen.

If R₁ is a substituted alkoxy group, and if the alkoxy group substituentis a dialkylamino group, the alkyl portion of the dialkylaminosubstituent group on the R1 alkoxy group preferably contains 1 to 6carbons, and more preferably 2 to 3 carbons, and most preferably is abeta-dialkylethoxy group. Examples of preferred dialkylaminoalkoxygroups include dimethylaminoethoxy and diethylaminoethoxy groups.

If R₁ is a substituted alkoxy group, and if the alkoxy group substituentis an alkylsulfonyloxy group, the alkyl portion of the alkylsulfonyloxysubstitutent group on the R1 alkoxy group preferably contains 1 to 6carbons, and more preferably 1 to 3 carbons. An example of a preferredalkylsulfonyloxy alkoxy group is methanesulfonyloxyethoxy.

If R₁ is a substituted alkoxy group, and if the alkoxy group substituentis a substituted or unsubstituted heterocycle containing between 5 or 6members, the heterocycle substituent preferably contains at least onenitrogen and more preferably 1 to 2 nitrogens. Preferred heterocyclesinclude piperidino and piperazino groups. Other saturated andunsaturated heterocycles containing nitrogen may also be used.

If the heterocycle substituent group on the R1 alkoxy group issubstituted, it preferably is substituted with an alkyl group. Thesubstituent alkyl group preferably contains from 1 to about 6 carbonatoms, and more preferably contains from 1 to 3 carbon atoms. Apreferred example of an alkyl substituted heterocycle alkoxy group is amethylpiperazinoethoxy group.

In the compounds of the present invention any of the foregoing groupsdescribed herein as being represented by R1 may also be at the R₂position provided that both R1 and R2 may not both be simultaneouslyhydrogen, simultaneously hydroxy, simultaneously methoxy orsimultaneously acetate. The R₁ and R₂ groups may be at any position onthe respective phenyl group; however, the para positions are preferred.

When the compound of the present invention is optically active, eachoptical isomer thereof, as well as racemic mixtures thereof of opticalisomers having the same chemical structures are within the scope of thepresent invention.

One particularly preferred compound of the present invention comprises(Z)-1,1-Dichloro-2-[4-(2-methanesulfonyloxyethoxy)phenyl]-3-phenylcyclopropane:##STR3## including each of its (R) and (S) optical isomers and racemicmixtures thereof.

Another preferred compound is(Z)-1,1-Dichloro-2-[4-[2-(dimethylamino)ethoxy]-phenyl]-3-phenylcyclopropane:##STR4## including each of its (R) and (S) optical isomers and racemicmixtures thereof.

Another preferred compound is(Z)-1,1-Dichloro-2-[4-[2-(diethylamino)ethoxy]-phenyl]-3-phenylcyclopropane:##STR5## including each of its (R) and (S) optical isomers and racemicmixtures thereof.

Another preferred compound is(Z)-1,1-Dichloro-2-[4-[2(piperidino)ethoxy]-phenyl]-3-phenylcyclopropane:##STR6## including each of its (R) and (S) optical isomers and racemicmixtures thereof.

Yet another preferred compound is(Z)-1,1-Dichloro-2-[4-[2-(N-methylpiperazino)ethoxy]-phenyl]-3-phenylcyclopropane:##STR7## including each of its (R) and (S) optical isomers and racemicmixtures thereof.

Preferably, the compounds of the present invention are combined with apharmaceutically acceptable carrier to form a pharmaceutical compositionappropriate for therapeutic delivery to a mammal. The pharmaceuticallyacceptable carrier should not substantially interfere with theanti-estrogenic and anti-tumor activities of the compound, and may be asolid or liquid in which the compound is solubilized, suspended ordispersed in any manner.

The compounds of the present invention may be administered orally insolid dosage forms, such as tablets and powders, or in liquid dosageforms, such as elixirs, syrups and suspensions; they may also beadministered parenterally, in sterile liquid dosage forms. Suchparenteral administration may include intravenous, intramuscular,subcutaneous, intra-arterial, and direct tumor perfusion techniques.

If the compound is to be injected, the pharmaceutical carrier shouldpreferably be isotonic, and have about a physiological pH. Suitablepharmaceutical carriers for parenteral administration may be anysuitable oil, saline, aqueous dextrose or related sugar solutions, orglycols such as propylene glycol or polyethylene glycols. Solutions forparenteral administration contain preferably a water soluble salt of theactive ingredient, suitable stabilizing agents, and if necessary, buffersubstances. Additionally, parenteral solutions can containpreservatives. Other suitable pharmaceutical carriers are described inRemington's Pharmaceutical Sciences, Mack Publishing Co., and similarreference texts.

The present invention further comprises a method of inducingantiestrogenic activity in a mammal, such as a human, in need of suchtherapy comprising administering to the mammal an antiestrogenicallyeffective amount of one or more compounds having the formula, preferablyin the form described above. The dosage of the compounds of the presentinvention may vary due to the therapeutically desired result which isaffected by the type of disease or condition in the mammal; the age,weight and health of the recipient; the severity of the condition ordisease in the mammal; the kind of concurrent treatment, if any, beingadministered to the mammal; and the frequency of treatment. Generally, atherapeutically effective dosage is less than about 0.5 mg to about 2 mgper kilogram of body weight of the mammal over a 24 hour period. Themethod of administration of the compound of the present invention can beby any suitable method as previously described.

The present invention also comprises a method of inhibiting thedevelopment of an estrogen-dependent tumor in a mammal, such as a human,in need of such therapy comprising administering to the mammal atherapeutically effective amount of one or more compounds having theformula described above, preferably in the form of a pharmaceuticalcomposition comprising at least one of the compounds combined with apharmaceutically acceptable carrier. "Inhibiting the development of anestrogen-dependent tumor" means either slowing the growth of a tumor,diminishing the size of a tumor, or preventing the formation of a tumorfrom cells having the potential of developing into a tumor wherein thetumor requires the presence of an estrogenic substance for the growth,development and/or metastatic involvement of the tumor.

The compounds previously described may be administered to the mammal toinhibit the development of the estrogen-dependent tumor by anadministration method of the type previously described. The dosage mayvary according to the type of the disease; the size of the tumor ortumors, if present; and the quantity of tumors as well as other factorspreviously described. Generally, a daily dosage of less than about 0.5mg to about 2 mg/kg of body weight of the mammal will suffice.

The following examples illustrate the practice of the present invention.

EXAMPLE 1 Preparation of(Z)-1,1-Dichloro-2-[4-(2-methanesulfonyloxyethoxy)phenyl]-3-phenylcyclopropane(Compound 7)

A mixture of p-hydroxybenzaldehyde (40.30 g, 0.33 mol), 2-chloroethylmethyl ether (31.20 g 0.33 mol) and K₂ CO₃ (45.61 g, 0.33 mol) in DMF(100 mL) was refluxed for 12 h. The resulting orange mixture was dilutedwith water (100 mL) and extracted three times with 50 mL of CHCl₃. Theorganic layers were combined and washed with 5% NaOH, brine and driedover anhydrous MgSO₄. It was filtered and evaporated in vacuo to givebrown liquid which was distilled on the Kugelrohr distillation apparatusto obtain yellowish liquid (51.40 g, 87%) at room temperature but solidin the cold. NMR (CDCl₃) δ 3.40 (s, 3H, OCH₃), 3.60-3.90 (m, 2H, OCH₂),4.10-4.40 (m, 2H, OCH₂), 7.00-7.80 (dd, 4H, substituted ArH), 9.90 (s,1H, CHO). This procedure produces 4-[2-(methoxy)ethoxy]-benzaldehyde.

To phenylacetic acid (66.12 g, 0.49 mol) was added acetic anhydride (75mL), the acid dissolved with continous stirring to give a light yellowsolution. Triethylamine (45 mL) was added,4-[2-(methoxy)ethoxy]benzaldehyde (58.34 g, 0.32 mol) was added slowlythrough a dropping funnel and the mixture refluxed for 36 h. The mixturewas allowed to cool to room temperature and transferred to a separatoryfunnel with 100 mL of ET₂ O. Four 40 mL portions of 30% NaOH solutionwas used to extract the ET₂ O mixture. The combined basic layers werewashed with two 50 mL portions of ET₂ O which were discarded. The basicsolution was then acidified with concentrated HCl to give a solidprecipitate which was recrystallized from 95% ethanol to give lightyellow needles (48.86 g, 52%), m.p. 154°-156° C. NMR (CDCl₃) δ 3.40 (s,3H OCH₃), 3.60-3.90 (m, 2 H, OCH₂), 4.00-4.20 (m, 2H, OCH₂), 6.70 and7.05 (dd, 4H, substituted ArH), 7.20-7.50 (m, 5H, ArH), 7.95 (s, 1H,C═CH). This procedure produces (Z)-α-Phenyl-β-[4-(2-methoxy)ethoxy]cinnamic acid.

A 500 mL, three necked, round bottom flask equipped with a condenser,with a gas trap, a thermometer and a magnetic stirrer was utilized. To asolution of (Z)-α-Phenyl-β-[4-(2-methoxy) ethoxy]cinnamic acid (20.00 g,0.07 mol) in quinoline (90.41 g, 0.70 mol) was added copper chromite(1.46 g, 0.005 mol) and heated. Carbon dioxide evolved when thetemperature reached 190° C. The mixture was kept within 200°-210° C. for3 h, cooled to room temperature and filtered under vacuum. The blackmixture was transferred to a separatory funnel with 100 mL of ET₂ O andextracted with five 50 mL portions of 20% HCl solution to remove theresidual quinoline. The ET₂ O layers were washed with brine, dried overMgSC₄ and concentrated in vacuo. The resulting brown oil was purified byflash chromatography (silica gel, 50/50 CH₂ Cl₂ / petroleum ether) toobtain cis stilbene 4 (15.61 g, 91%) as a very light yellow oil. NMR(CDCl₃) δ 3.40 (s, 3H, OCH₃), 3.60-3.90 (m, 2H, OCH₂), 4.00-4.20 (m, 2H,OCH₂), 6.55 (s, 2H, C═CH), 6.80 and 7.20 (dd, 4H, substituted ArH), 7.30(s, 5H, ArH). This procedure produces(Z)-1-[4-(2-methoxyethoxy)phenyl]-2-phenylethylene.

To (Z)-1-[4-(2-methoxyethoxy)phenyl]-2-phenylethylene (15.61 g, 0.06mol) dissolved in CHCl₃ (193 mL) was added triethylbenzyl-ammoniumchloride (0.98 g, 0.004 mol), chilled 40% NaOH solution (96.00 g, 2.40mol) was added slowly through a dropping funnel according to the methodof Dehmlow and Schonefeld, J. Liebiqs Ann Chem. 744:42 (1971) and themixture stirred for 80 h. The resulting brown emulsion was poured onto200 mL water in a separatory funnel and the layers were separated, theaqueous layer was extracted with three 50 mL portions of CH₂ Cl₂, thecombined organic extracts were washed with brine, dried over MgSO₄,filtered and evaporated in vacuo to obtain a dark brown oil which waspurified by flash chromatography (50:50 CH₂ Cl₂ /petroleum ether) togive a colorless oil (19.70 g, 80%). NMR (CDCl₃) δ 3.30 (s, 2H, ArCH),3.50 (s, 3H, OCH₃), 3.65-3.85 (m, 2H, OCH₂), 4.00-4.25 (m, 2H, OCH₂),6.85-7.40 (m, 9H, ArH). This procedure produces(Z)-1,1-Dichloro-2-[4-(2-methoxyethoxy)phenyl]-3-phenylcyclopropane.

To (Z)-1,1-Dichloro-2-[4-(2-methoxyethoxy)phenyl]-3-phenylcyclopropane(14.10 g, 0.04 mol) in CH₃ CN (100 mL) was added sodium iodide (25.07 g,0.17 g) . The reaction flask was fitted with a gas trap, an argon inletand a rubber septum. The reaction flask was then flushed with argon. Bymeans of a syringe, chlorotrimethylsilane (21 mL) was injected into thereaction flask through the rubber septum and the mixture stirred at roomtemperature for 24 hours. Water (100 mL) was then added to the mixtureand extracted with three 50 mL portions of ethyl acetate. The organicextracts were combined and washed with sodium thiosulfate solution tillthe organic extract became colorless, then dried over anhydrous MgSO₄and evaporated in vacuo to obtain an oil. The oil was purified by flashchromatography and eluted with CH₂ Cl₂ to give a colorless oil (10.79 g,80%). NMR (CDCl₃) δ 2.20 (broad, 1H, OH), 3.30 (s, 2H, ArCH), 3.90-4.20(m, 4H, OCH₂ CH₂ OH), 6.80-7.50 (m, 9H, ArH). This procedure produces(Z)-1,1-Dichloro-2-[4-(2-hydroxy-ethoxy)-pheny]-3-phenylcyclopropane.

Triethylamine (14.87 g, 0.15 mol) was added to a solution(Z)-1,1-Dichloro-2-[4-(2-hydroxyethoxy)-pheny]-3-phenylcyclopropane(10.78 g, 0.03 mol) and methanesulfonyl chloride (16.81 g, 0.15 mol) inTHF (50 mL) at 0° C. After 1 h water (50 mL) was added to the reactionmixture and transferred to a separatory funnel. The mixture wasextracted three times with 30 mL portions of ethyl acetate, dried overMgSO₄ and concentrated in vacuo to give a light yellow oil. The oil waspurified by flash chromatography (50:50 CH₂ Cl₂ /petroleum ether) togive a colorless oil (11.65 g, 87%). NMR (CDCl₃) δ 3.10 (s, 3H, OSO₂CH₃), 3.30 (s, 2H, ArCH), 4.10-4.30 (m, 2H, OCH₂ OSO₂ CH₃), 4.50-4.70(m, 2H, ArOCH₂), 6.80-7.40 (m, 9H, ArH).

EXAMPLE 2 Preparation of(Z)-1,1-Dichloro-2-[4-[2-(dimethylamino)ethoxy]-phenyl]-3-phenylcyclopropane(compound 8)

To the(Z)-1,1-Dichloro-2-[4-(2-methanesulfonyloxyethoxy)phenyl]-3-phenylcyclopropane(1.00 g, 0.003 mol) in 25 mL CH₃ CN was added dimethylaminehydrochloride (2.45 g, 0.03 mol) and the mixture cooled in dryice/acetone bath for 25 min. Flame dried K₂ CO₃ (8.29 g, 0.06 mol) wasthen added and the flask stoppered with a rubber septum so as to trapthe gaseous dimethylamine. The slurry mixture was stirred at roomtemperature for 48 h after which 25 mL of water was added and extractedwith three portions of 10 mL ET₂ O. The ET₂ O extracts were combined,dried over MgSO₄ and evaporated in vacuo, the residue was purified byflash chromatography and eluted with EtOAc to give a light yellowishoil. The oil was dissolved in 25 mL Et₂ O and extracted with 10 mLportions of 30% HCl solution until the ET₂ O layer became clear. Thecombined aqueous extracts were treated with 20% NaOH solution to pH 11,and extracted with three portions of 20 mL ET₂ O. The combined ET₂ Oextracts were washed with water, dried over MgSO₄ and concentrated invacuo to obtain a light yellow oil (0.60 g, 71%). NMR (CDCl₃) δ 2.35 (s,6H, N(CH₃)₂), 2.70 (t, 2H, CH₂ N), 3.30 (s, 2H, ArCH), 4.05 (t, 2H,OCH₂), 6.90-7.40 (m, 9H, ArH).

Citrate Salt: The oil(Z)-1,1-Dichloro-2-[4-[2-(dimethylamino)ethoxy]-phenyl]-3-phenylcyclopropanewas dissolved in hot EtOH and treated with an equal molar amount ofcitric acid in hot EtOH. The citrate salt was recrystallized from EtOHand obtained as a white powder in 81% yield, mp: 67°-68° C.

EXAMPLE 3 Preparation of(Z)-1,1-Dichloro-2-[4-[2-(diethylamino)ethoxy]-phenyl]-3-phenylcyclopropane(compound 9)

Diethylamine (10 mL) and Et₃ N (10 mL) were added to a solution of(Z)-1,1-Dichloro-2-[4-(2-methanesulfonyloxyethoxy)phenyl]-3-phenylcyclopropane(1.00 g, 0.003 mol) in acetonitrile (20 mL). The orange solution wasstirred at room temperature for 48 h after which the volatile componentswere evaporated in vacuo and the residue purified by flashchromatography eluting with EtOAc to give an orange oil. Furtherpurification was carried out by acid/base extraction as in the procedurefor(Z)-1,1-Dichloro-2-[4-[2-(dimethylamino)ethoxy]-phenyl]-3-phenylcyclopropaneto obtain a light orange oil (0.66 g, 70%) NMR (CDCl₃) δ 1.15 (t, 6H,NCH₂ CH₃), 2.50-3.00 (m, 6H, OCH₂ CH₂ and NCH₂ CH₃), 3.30 (s, 2H, ArCH),4.1O (t, 2H, OCH₂), 6.90-7.40 (m, 9H, ArH).

Citrate Salt: The oil(Z)-1,1-Dichloro-2-[4-[2-(diethylamino)ethoxy]-phenyl]-3-phenylcyclopropanewas dissolved in hot EtOH and treated with an equal amount of citricacid in hot EtOH. The citrate salt was recrystallized from EtOH andobtained as a white powder in 83% yield, mp: 101°-102° C.

EXAMPLE 4 Preparation of(Z)-1,1-Dichloro-2-[4-[2-(piperidino)ethoxy]-phenyl]-3-phenylcyclopropane(compound 10)

This compound was prepared according to the procedure used to synthesize(Z)-1,1-Dichloro-2-[4-[2-(diethylamino)ethoxy]phenyl]-3-phenylcyclopropanedescribed herein using (Z)-1,1-Dichloro-2-[4-(2-methanesulfonyloxyethoxy)phenyl]-3-phenylcyclopropane(1.00 g, 0.003 mol) and piperidine (10 mL). The compound was obtained asa light orange oil (0.84 g, 87%). NMR (CDCl₃) δ 1.40-1.70 (m, 6H, ringCH₂ nonadjacent to N), 2.40-2.60 (broad m, 4H, CH₂ adjacent to N), 2.75(t, 2H, CH₂ N), 3.30 (s, 2H, ArH), 4.10 (t, 2H,OCH₂), 6.80-7.40 (broadm, 9H, ArH).

Citrate Salt: The oil(Z)-1,1-(Dichloro-2-[4-[2-(piperidino)ethoxy]-phenyl]-3-phenylcyclopropanewas dissolved in hot EtOH and treated with an equal amount of citricacid in hot EtOH. The citrate salt was recrystallized from EtOH andobtained as a white powder in 85% yield, mp: 96°-100° C.

EXAMPLE 5 Preparation of(Z)-1,1-Dichloro-2-[4-[2-(N-methylpiperazino)ethoxy]-phenyl]-3phenylcyclopropane (compound 11)

This compound was prepared according to the procedure used to synthesize(Z)-1,1-Dichloro-2-[4-(2-diethylamino)ethoxy]-phenyl]-3-phenylcyclopropanedescribed herein using(Z)-1,1-Dichloro-2-[4(2-methanesulfonyloxyethoxy)phenyl-3-phenylcyclopropane(1.00 g, 0.003 mol) and N-methyl piperazine (10 mL). The product wasobtained as a colorless oil (0.82 g, 81%). NMR (CDCl₃) δ 2.27 (s, 3H,NCH₃), 2.39-2.70 (broad m, 8H, ring CH₂), 2.80 (t, 2H, CH₂ N), 3.30 (s,2H, ArCH), 4,05 (t, 2H, OCH₂), 6.90-7.40 (m, 9H, ArH). Citrate Salt: Theoil (Z)-1,1-Dichloro-2-[4-[2-(Nmethylpiperazino)ethoxy]-phenyl]-3-phenylcyclopropane was dissolved inhot EtOH and treated with an equal amount of citric acid in hot EtOH.The citrate salt was recrystallized from EtOH and obtained as a whitepowder in 90% yield, mp: 101°-102° C.

EXAMPLE 6 Biological Testing

The biological evaluation of the test compounds consisted of the invitro rat cytosolic estradiol receptor binding assay, the in vivoimmature mouse uterotrophic (estrogenic) assay, and the in vivo immaturemouse and rat antiuterotrophic (antiestrogenic) assay and in the invitro suppression of the proliferation of the MCF-7 human breast cancercell line. All assays contained estradiol, TAM, MER 25, and1-1-dichloro-2-3 -cis-diphenylcyclopropane (Analog II) as standards.

Biological Assays. Tamoxifen was obtained from Stuart Pharmaceutical,Division of ICI Americas, Inc., Wilmington, Del. MER 25 was obtainedfrom Merrell Dow Research Institute, Division of Merrell DowPharmaceuticals, Inc., Cincinnati, Ohio. Absolute ethanol was obtainedfrom U.S. Industrial Chemicals Co. Hormones and biochemicals werepurchased from Sigma Chemical Co. Animals and Housing. Viral-freeimmature female Swiss-Webster mice were obtained at 17-19 days of agefrom Sasco (Omaha, Nebr.) weighing 8-10 g, and were used in theuterotrophic and antiuterotrophic assays. Immature female Sprague-Dawleyrats, obtained also at 17-19 days of age from Sasco, weighing 28-33 g,were used in the estradiol receptor binding assay. Animals were housedin wire topped polycarbonate cages with six animals per cage.Environment was controlled at 25° C. with a 12-hour light/dark cycle.The animals received a diet of Wayne Lab Blox rodent chow and tab waterad libitum.

EXAMPLE 7 Uterotrophic Assay

Estrogenic activity of the compounds was determined using a modificationof the method of Rubin, B. L., et al., Endocrinology 49: 429 (1951) (seePento, J.T., et al.;l J. Endocrinol. 61: 1216 (1978)) using immature(17-19 days old) female Swiss-Webster mice. The test compounds weredissolved separately in a minimum amount of isopropyl myristate (IPM),and diluted serially with sesame oil to the proper concentrations (finalconcentration of IPM<5%). Solutions were shaken at 25° C. for severalhours to ensure complete dissolution. The mice were randomly separatedinto groups of six animals, weighed, and the compounds were administeredby s.c. injection of 0.1 mL of the oil solutions into the nape of theneck for 3 consecutive days. The solutions were periodically checked byTLC to insure homogeneity. A control group received 0.1 mL sesame oilalone.

The animals were anesthetized with ET₂ O and sacrificed by cervicaldislocation 24 h after the last injection. Body weights were determinedand the uteri were removed, cleaned of adhering connective tissue andfat, blotted to remove tissue fluid, and weighed to the nearest 0.1 mg.

The compounds shown in Table I, with the exception of compound 11, didnot produce any uterotrophic activity in the immature mouse at doses of30, 150, and 750 μg. Compounds 7, 8, 9 and 10 caused a reduction inuterine weight as compared with MER25 which produced estrogenicresponses at the 150 and 750 μg doses.

                  TABLE I                                                         ______________________________________                                        (Z)-1,1-Dichloro-2,3-Diarylcyclopropane Derivatives                           Compound                                                                      No.        R                Formula                                           ______________________________________                                        7          OSO.sub.2 CH.sub.3                                                                             C.sub.18 H.sub.18 Cl.sub.2 SO.sub.4               8          N(CH.sub.3).sub.2                                                                              C.sub.19 H.sub.21 Cl.sub.2 NO                     9          N(CH.sub.2 CH.sub.3).sub.2                                                                     C.sub.21 H.sub.25 Cl.sub.2 NO                     10         c-NC.sub.5 H.sub.10                                                                            C.sub.22 H.sub.25 Cl.sub.2 NO                     11         c-N(CH.sub.2 CH.sub.2).sub.2 N--CH.sub.3                                                       C.sub.22 H.sub.26 Cl.sub.2 NO                     ______________________________________                                    

EXAMPLE 8

Antiuterotrophic Assay

Antiestrogenic activity of the compounds was determined by inhibition ofthe estradiol-induced uterotrophic activity in immature femaleSwiss-Webster mice. Animals were distributed into groups of six animals.A modification of the uterotrophic assay described in Example 21 wasused (Dorfman, R. I., et al., Endocrinology 68: 17 (1960)). Estradiolwas dissolved in sesame oil (0.1 μg/mL). The test compounds weredissolved in IPM and diluted with IPM to achieve desired concentrations.The solutions were periodically checked by TLC to insure homogeneity.Injections were made in the nape of the neck for 3 consecutive days. Theunstimulated control group received vehicles alone (0.05 mL IPM and 0.1mL sesame oil each day), while the stimulated control group received 0.1mL of the estradiol solution (total dose 0.03 μg). All test groupsreceived 0.1 mL of the stimulating dose of estradiol (0.01 μg) plus 0.05mL of the test compounds solutions each day. The IPM and oil injectionswere made at separate sites to minimize possible physical or chemicalinteractions or reduced absorption of either compound. Antiestrogenicactivity was measured as a decrease from the estradiol-induced increasein uterine weight seen in the test compound groups versus theestradiol-stimulated group alone.

A series of side-chain derivatives of the antiestrogen, Analog II andMER 25 were examined for estrogen antagonism at doses of 30, 150 and 750μg against a stimulating dose of 0.03 μg of estradiol.

Compounds 8, 9, 10 and 11 produced some antiestrogenic activity, whilecompound 7 did not (Table II). Compound 9 induced an antiestrogenresponse of 39.7% as compared to 37.1% of Analog II at 30 and 750 μgdosages respectively. The compounds in Table I did not produce anysignificant decrease in uterine weight at the highest dose of 750 μg butelicited antiestrogenic activity at the lower doses of 30 and 150 μg.

Analog II and MER 25 produced a dose dependent decrease in uterineweight, with MER 25 eliciting the highest observed antiestrogenicactivity of 66%. None of the compounds potentiated the uterine weightgain from the stimulating dose of 0.03 μg of estradiol.

                  TABLE II                                                        ______________________________________                                        Observed Antiestrogenic Activities.sup.a                                                 Total Dose                                                                              % Observed Antiestrogenic                                Compound   (μg)   Activity.sup.aa                                          ______________________________________                                        7           30        7.23                                                               150       --                                                                  750       --                                                       8           30        9.26                                                               150       25.20                                                               750       11.25                                                    9           30       39.70                                                               150       22.44                                                               750       15.50                                                    10          30       28.41                                                               150       33.87                                                               750       27.79                                                    11          30       10.15                                                               150       32.10                                                               750       17.53                                                    Analog II   30       24.65                                                               150       31.96                                                               750       37.12                                                    MER-25      30       24.46                                                               150       24.17                                                               750       66.42                                                    ______________________________________                                         .sup.a Determined as the decrease in the estradiolstimulated (0.03 μg      total dose) uterine wt. of immature female mice.                              .sup.aa Calculated by: {(mean uterine wt. of estradiolstimulated  mean        uterine wt. of control)  (mean uterine wt. of test compounds  mean uterin     wt. of control)/(mean uterine wt. of estradiolstimulated  mean uterine wt     of control)} × 100.                                                

EXAMPLE 9 Receptor Binding Assay

The receptor binding activies of the test compounds for the estrogenicreceptors were determined by displacement of [³ H] estradiol from ratuterine cytosol in vitro. Female Sprague-Dawley rats (17-19 days old)were treated with 0.53 μg of estradiol in 0.1 mL sesame oil for threeconsecutive days (total dose 1.6 μg). On the fourth day the rats wereanesthetized with ET₂ O and sacrificed by cervical dislocation. Amodification of Korenman's receptor binding assay method (Korenman,S.G., Steroids 13: 163 (1969)) was used. Uteri were removed, cleaned ofadhering connective tissue and fat, weighed (avg. wt.=83 mg/animal), andhomogenized (Polytron PT-10 stainless steel homogenizer, rheostatsetting 7, five ten-second bursts with a ten-second pause betweenbursts) at 0°-4° C. in five volumes (w/v) of TEDM buffer (10 mMTris-HCl, 1.5 mM disodium ethylenediamine tetraacetic acid, 1.0 mMdithiothreitol, 10.0 mM sodium molybdate, pH adjusted to 7.4 with 5MNaOH). The resulting homogenate was centrifuged at 2000 g for 15 min (4°C.). The supernatant was then centrifuged at 104,000 g for 1 h (4° C.).The supernatant from the high speed centrifugation (cytosol) wascarefully decanted and used immediately. The protein content of thecytosol was determined and adjusted to 4-5 mg protein/mL. The testcompounds were dissolved in EtOH or DMSO and diluted with TEDM so thatthe final EtOH concentration was less than 2% or the final DMSOconcentration was less than 10%. Neither of these concentrations of theorganic solvents affected the binding of the tritiated estradiol to thecytosolic receptor or the amount of non-specific binding seen asdetermined by parallel incubations. Duplicate incubations were conductedat 4° C. for 24 h in a total volume of 0.5 mL containing: 200 μLcytosol; 100 μL (0.218 μCi) of 2,4,6, 7(n)-[³ H]-17β-estradiol (93.35mCi/mmole); 100 μL of the test compounds at concentrations ranging from10⁻⁴ to 10⁻⁶ M, or unlabelled estradiol at concentrations ranging from10⁻⁶ to 10⁻⁸ M; and sufficient TEDM to obtain a final volume of 0.5 mL.Single parallel incubations at each concentration of test compound andestradiol contained 100 μL of 2×10⁻⁵ M DES in the final TEDM addition todistinguish between specific receptor binding and nonspecificprotein/receptor binding of the compounds.

After incubation, 0.5 mL of a Dextran-Coated Charcoal (DCC) solution(0.5% activated charcoal and 0.05% Dextran T-70, w/v, in TEDM buffer)was added and the tubes were gently vortexed at 4° C. 15 min. The tubeswere centrifuged at 2000 g 15 min (4° C.) to remove the unbound [³H]-estradiol. A 0.5 mL aliquot of the supernatant was added to 10 mLBeckmann Ready-Solv VI scintillation cocktail in subdued light and thetritium content of each vial was determined by liquid scintillationspectrometry. The radioactivity was plotted as a function of the logconcentration of competing ligand and subjected to linear regressionanalysis. Relative binding affinity of each compound was determined bythe method of Bliss, C.I., The Statistics of Bioassay, Academic Press(New York 1952).

The compounds in Table I produced no displacement of [³ H]-estradiolfrom the rat uterine cytosol, while tamoxifen and Analog II produced aparallel displacement with estradiol indicating a relative bindingaffinity for the ER. Both the free bases and the citrate salts of thecompounds were tested in the ER binding assay.

EXAMPLE 10

Statistical Analysis

The relative binding affinity determinations were performed by a TI-59programmable calculator (Texas Instruments). The Student's t-test(non-paired) was used to compare individual treatment groups to theestradiol group statistically. Multiple group comparison were analyzedby ANOVA. P or F values of less than 0.05 were considered to besignificant. The Student's t values, ANOVA F values, linear regressionand standard errors were obtained by Cricket Graph and StatWorksprograms on a MacIntosh computer.

EXAMPLE 11

The compounds shown in Table I were found to be devoid of estrogenicactivity, but possessed a small degree of antiestrogenic activity in themouse. These compounds produced a 10 to 37% decrease inestradiol-stimulated uterotropic activity at the doses tested (see TableII). None of the cyclopropyl side-chain compounds (Table I) displaced [³]-estradiol from the ER in the rat uterine cytosol as determined by thecompetitive binding assay. Since it is known that there is a correlationbetween ER binding affinity and estrogenic activity, it can be assumedfrom the receptor binding data that the compounds, which were poorbinders, lacked estrogenic activity. As observed in the uterotrophicassay, none of the compounds (Table I) increased uterine weight ascompared to the non-treated control group and they did not potentiatethe uterotrophic response of the stimulating dose of estradiol in theantiuterotrophic assay, clearly indicating that the compounds are notestrogenic in the mouse. Analog II and MER-25 used as standards,elicited a significant decrease in uterine weight only at the highesttested dose of 750 μg. The structure of Analog II is as follows:##STR8##

EXAMPLE 12

In Vitro testing of MCF-7, Human Breast Cell Cancer Line

MCF-7 Cell Culture Method. MCF-7 human breast cancer cells were obtainedfrom the Michigan Cancer Foundation. MCF-7cells were grown at 37° C. in75 cm² tissue culture (T-75) flasks, as monolayer cultures in RPMI 1640media (without phenol red) supplemented with 2 mM 1-glutamine,gentamicin (50 μg/mL), penicillin (100 units/mL), streptomycin (100μg/mL) and calf serum (5%). Cultures were grown in an incubator at 37°C. in a humid 5% CO₂ atmosphere, and fed on alternate days. Exponentialgrowth was maintained by subculturing at eight day intervals when thecell number per T-75 flask reached 10 to 12×10⁶ cells.

All of the compounds were tested for their ability to alter the growthof MCF-7 cells at a concentration ranging from 10⁻⁴ to 10⁻⁶ M. Growthinhibition produced at concentrations greater than 5×10⁻⁶ M areconsidered to be cytotoxic anti-tumor effects. Control samples receivedvehicle alone at the same concentrations used in the treatment groups.

In each experiment, the cells were trypsinized, washed and plated inmultiwell plates at a density of 5×10⁴ cells/well in 3 mL of RPMI 1640media. Cells were allowed to attach and were in logarithmic growth whenthe test compounds were added. Each group was done in triplicate. Thetest compounds were dissolved in a polyethylene glycol 400:ethanol(55:45) mixture and added in the culture media. The final concentrationof the vehicle mixture was 0.1% of the incubation media. Cell growth wasmeasured on alternate days using the hemocytometric trypan blueexclusion method.

Statistics. The student's t-test (non-paired) was used to makestatistical comparisons between two experimental groups. Multiple groupcomparisons in the cell culture experiments were made using a 3-wayANOVA. P values of less than 0.05 were considered to be statisticallysignificant. The student's t values, standard errors, and linearregression were obtained using the Stat Work and Cricket Graph programson a Macintosh computer.

Results. The results of the MCF-7 human breast cancer cell culture studyindicate that compound 10 abolished cell growth at 10⁻⁴ M (Table III),significantly reduced cell growth at 10⁻⁵ M (Table IV), and had noeffect on cell growth at 10⁻⁶ M (Table V). These data indicate thatcompound 10 is cytotoxic to human breast cancer cells in culture. Cellgrowth was inhibited by tamoxifen (a known antiestrogen) at aconcentration of 10⁻⁶ M confirming the estrogen-specific nature of theMCF-7 cell used in this study.

                  TABLE III                                                       ______________________________________                                        Effect of 10.sup.-4 M Compound 10 on Cell Growth                              Mean Live Cell Count Per Well                                                 Days Control   Compound 10, 10.sup.-4 M                                                                      Tamoxifen, 10.sup.-6 M                         ______________________________________                                        0     81,481   81,481          --                                             2    158,333   --              --                                             4    256,944   5,324           --                                             6    235,417   1,620           --                                             ______________________________________                                    

                  TABLE IV                                                        ______________________________________                                        Effect of 10.sup.-5 M Compound 10 on Cell Growth                              Mean Live Cell Count Per Well                                                 Days Control   Compound 10, 10.sup.-5 M                                                                      Tamoxifen, 10.sup.-6 M                         ______________________________________                                        0    118,056   118,056         118,056                                        2    227,315   115,741         248,611                                        4    474,074   186,111         332,407                                        6    648,148    50,000         547,222                                        ______________________________________                                    

                  TABLE V                                                         ______________________________________                                        Effect of 10.sup.-6 M Compound 10 on Cell Growth                              Mean Live Cell Count Per Well                                                 Days Control   Compound 10, 10.sup.-6 M                                                                      Tamoxifen, 10.sup.-6 M                         ______________________________________                                        0     91,204    91,204          91,204                                        2    149,537   215,278         105,093                                        4    415,741   430,556         387,963                                        6    526,389   537,037         647,222                                        ______________________________________                                    

All U. S. patent applications and publications cited herein are herebyincorporated by reference.

Changes may be made in the embodiments of the invention described hereinor in parts or elements of the embodiments described herein or in thesteps or in the sequence of steps of the methods described hereinwithout departing from the spirit and scope of the invention as definedin the following claims.

What is claimed is:
 1. A compound having the formula: ##STR9## or any pharmaceutically acceptable salt thereof, in which: X is selected from a group consisting of,halogen atoms and hydrogen atoms; R₁ is selected from a group consisting of,a hydrogen atom, and a substituted alkoxy group in which the substituent of the alkoxy group is selected from a group consisting of,an unsubstituted piperazine, and a substituted piperazine in which the substituent is an alkyl group containing from 1 to 6 carbon atoms; and R₂ is selected from a group consisting of,a hydrogen atom, and a substituted alkoxy group in which the substituent of the alkoxy group is selected from a group consisting of, an unsubstituted piperazine, and a substituted piperazine in which the substituent is an alkyl group containing from 1 to 6 carbon atoms; and with the proviso that R1 and R2 cannot be simultaneously hydrogen.
 2. The compound of claim 1 in which R₁ is selected from a group consisting of:a substituted alkoxy in which the substituent is piperazine; and a substituted alkoxy in which the substituent is a substituted piperazine and in which the piperazine substituent is an alkyl group.
 3. The compound of claim 2 in which R₂ is hydrogen.
 4. The compound of claim 3 in which R₁ and the R₂ are each at the para ring position.
 5. The compound of claim 4 in which X is chlorine.
 6. The compound of claim 1 in which R₂ is selected from a group consisting of:a substituted alkoxy in which the substituent is piperazine; and a substituted alkoxy in which the substituent is a substituted piperazine and in which the piperazine substituent is an alkyl group.
 7. The compound of claim 6 in which R₁ is hydrogen.
 8. The compound of claim 7 in which the R₁ and the R₂ are each at the para ring position.
 9. The compound of claim 7 in which X is chlorine.
 10. The compound of claim 1 in which R₁ is a piperazionoethoxy group at the para ring position, R₂ is hydrogen and X is chlorine.
 11. The compound of claim 1 in which R₂ is a piperazionoethoxy group at the para ring position, R₁ is hydrogen and X is chlorine.
 12. A composition of matter comprising a compound having the formula: ##STR10## or any pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier of sufficient quantity to solubilize the compound, in which:X is selected from a group consisting of,halogen atoms and hydrogen atoms; R₁ is selected from a group consisting of,a hydrogen atom, a substituted alkoxy group in which the substituent of the alkoxy group is selected from a group consisting of, an unsubstituted piperazine, and a substituted piperazine in which the substituent is an alkyl group containing from 1 to 6 carbon atoms; and R₂ is selected from a group consisting of,a hydrogen atom, a substituted alkoxy group in which the substituent of the alkoxy group is selected from a group consisting of, an unsubstituted piperazine, and a substituted piperazine in which the substituent is an alkyl group containing from 1 to 6 carbon atoms; and with the proviso that R1 and R2 cannot be simultaneously hydrogen.
 13. The compound of claim 12 in which R₁ is selected from a group containing:a substituted alkoxy in which the substituent is piperazine; and a substituted alkoxy in which the substituent is a substituted piperazine and in which the piperazine substituent is an alkyl group.
 14. The compound of claim 13 in which R₂ is hydrogen.
 15. The compound of claim 14 in which R₁ and the R2 are each at the para ring position.
 16. The compound of claim 15 in which X is chlorine.
 17. The compound of claim 12 in which R₂ is selected from a group consisting of:a substituted alkoxy in which the substituent is piperazine; and a substituted alkoxy in which the substituent is a substituted piperazine and in which the piperazine substituent is an alkyl group.
 18. The compound of claim 17 in which R1 is hydrogen.
 19. The compound of claim 17 in which R₁ and the R₂ are each at the para ring position.
 20. The compound of claim 18 in which X is chlorine.
 21. A method of inducing antiestrogenic activity in a mammal in need of such therapy comprising administering to the mammal an antiestrogenically effective amount of one or more compounds having the formula: ##STR11## or any pharmaceutically acceptable salt thereof, in which: X is selected from a group consisting of,halogen atoms and hydrogen atoms; R₁ is selected from a group consisting of,a hydrogen atom, a substituted alkoxy group in which the substituent of the alkoxy group is selected from a group consisting of,an unsubstituted piperazine, and a substituted piperazine in which the substituent is an alkyl group containing from 1 to 6 carbon atoms; and R₂ is selected from a group consisting of,a hydrogen atom, a substituted alkoxy group in which the substituent of the alkoxy group is selected from a group consisting of, an unsubstituted piperazine, and a substituted piperazine in which the substituent is an alkyl group containing from 1 to 6 carbon atoms, and with the proviso that R1 and R2 cannot be simultaneously hydrogen.
 22. The method of claim 21 in which R₁ is selected from a group consisting of:a substituted alkoxy in which the substituent is a substituted piperazine; and in which the piperazine substituent is an alkyl group.
 23. The method of claim 22 in which R₂ is hydrogen.
 24. The method of claim 23 in which R₁ and the R₂ are each at the para ring position.
 25. The method of claim 24 in which X is chlorine.
 26. The method of claim 21 in which R₂ is selected from a group consisting of:a substituted alkoxy in which the substituent is piperazine, and a substituted alkoxy in which the substituent is a substituted piperazine and in which the piperazine substituent is an alkyl group.
 27. The compound of claim 26 in which R1 is hydrogen.
 28. The compound of claim 27 in which the R₁ and the R₂ are each at the para ring position.
 29. The compound of claim 28 in which X is chlorine.
 30. A method of inhibiting the development of an estrogen-dependent tumor in a mammal in need of such therapy comprising administering to the mammal an effective amount of one or more compounds having the formula: ##STR12## or any pharmaceutically acceptable salt thereof, in which: X is selected from a group consisting of,halogen atoms and hydrogen atoms; R₁ is selected from a group consisting of,a hydrogen atom,a substituted alkoxy group in which the substituent of the alkoxy group is selected from a group consisting of, an unsubstituted piperazine, and a substituted piperazine in which the substituent is an alkyl group containing from 1 to 6 carbon atoms; and R₂ is selected from a group consisting of,a hydrogen atom,a substituted alkoxy group in which the substituent of the alkoxy group is selected from a group consisting of, an unsubstituted piperazine, and a substituted piperazine in which the substituent is an alkyl group containing from 1 to 6 carbon atoms; and with the proviso that R1 and R2 cannot be simultaneously hydrogen.
 31. The method of claim 30 in which R₁ is selected from a group consisting of:a substituted alkoxy in which the substituent is piperazine; and a substituted alkoxy in which the substituent is a substituted piperazine and in which the piperazine substituent is an alkyl group.
 32. The method of claim 31 in which R2 is hydrogen.
 33. The method of claim 32 in which R₁ and the R₂ are each at the para ring position.
 34. The method of claim 33 in which X is chlorine.
 35. The of method claim 30 in which R₂ is selected from a group consisting of:a substituted alkoxy in which the substituent is piperazine; and a substituted alkoxy in which the substituent is a substituted piperazine and in which the piperazine substituent is an alkyl group.
 36. The compound of claim 35 in which R1 is hydrogen.
 37. The compound of claim 36 in which the R₁ and the R₂ are each at the para ring position.
 38. The compound of claim 37 in which X is chlorine. 